Diagnosis of breast implant associated anaplastic large cell lymphoma by analysis of cytokines in peri‐implant seromas
Authors: Honghua Hu, Elena Shklovskaya, Anand Deva, Haiying Xu, Kelly Fan, Kristen Brosamer, Richard Willson, Imran Khan, Mithun Sinha, Marshall E. Kadin
Journal: American Journal of Hematology (2023)

More than 1300 women with breast implants have developed an anaplastic large cell lymphoma (ALCL) in fluid (seroma) around their implant. More often, seromas are due to benign causes, for example, capsule contracture, leakage, or trauma. Our report in American Journal of Hematology identified several cytokines (IL-9, IL-10, IL-13) as significantly elevated only in seromas due to ALCL. We further showed that the most robust biomarker, IL-10, could be detected by a lateral flow assay (similar to COVID detection) within minutes allowing physicians to quickly plan management, eliminate or reduce costly testing and patient time away from family. Early detection of ALCL in seromas before infiltration may avoid need for cytotoxic or immunotherapy and is possibly life-saving. Breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) is a T-cell lymphoma of unknown pathogenesis that commonly presents as a delayed accumulation of fluid (seroma) around breast implants. To better understand the pathogenesis and clinical presentation of BIA-ALCL we analyzed the cytokines in seromas and capsule surrounding breast implants. BIA-ALCL is recognized by the World Health Organization as a lymphoma associated with breast implants of women after reconstructive surgery for breast cancer, prophylactic mastectomy because of high genetic risk for breast cancer, or cosmetic reasons.1 There are over 20 million women living with breast implants who have an estimated 1 in 3000 risk of developing BIA-ALCL.2 About 80% of patients present with breast swelling, pain and/or erythema associated with a seroma around the capsule of a textured surface breast implant a median time of 9 years after implant placement.3 The remaining patients develop infiltrative disease forming tumors in breast parenchyma, chest wall and/or regional lymph nodes requiring radiation, chemotherapy or immunotherapy.4 Patient survival is significantly improved by detection of BIA-ALCL when remaining localized to seromas and/or lining of the peri-implant capsule.5 Thus it is important to detect disease in early stages. To address the need for early detection, we performed analysis of cytokines in seromas collected from 45 Australian patients at Macquarie University Medical School in Sydney AU and 16 US patients at Rhode Island Hospital. Informed consent and Institutional approvals were obtained. All patients with seromas were Caucasian women who had textured breast implants. Based on morphological and immunohistochemical analyses, seromas were diagnosed as malignant (27 BIA-ALCL samples) or benign (34 samples including 10 contralateral samples). All but 3 BIA-ALCL patients presented with stage IA disease; 2 had stage IIA and one had disease recurrence. Seromas were centrifuged, and supernatants stored in plastic tubes at −80°C. Cell pellets were used for cytology and preparation of cell blocks for histology. BIA-ALCL seromas were characterized by large anaplastic tumor cells and apoptotic figures representing dead tumor cells focally attached to or infiltrating the capsule. Benign seromas contained a spectrum of small to medium-sized lymphoid cells with variable numbers of neutrophils, eosinophils, and histiocyte/macrophages (Figure S1). Tumor cells were shown to contain IL-10, IL-13, and IL-9 by immunohistochemistry (Figure S2). Seromas were analyzed by flow cytometry to measure the concentration of cytokines IL-2, 4, 5, 6, 9, 10, 13, 17A, 17F, 22, IFNγ, and TNFα using the LEGENDplex Human 12-plex T helper (Th) Cytokine Panel (Cat No 741028; BioLegend, San Diego, CA). For samples with saturating cytokine concentrations, repeat analyses were performed with higher dilutions (×4, ×10, ×30) until a reading in the linear range was obtained for all but one sample. Results showed that mean levels of IL-9, IL-10, and IL-13 were elevated 279-, 671-, and 20-fold, respectively, in BIA-ALCL compared to benign effusions, and each cytokine separated the two groups with little overlap (p < .0001) (Figure 1A). In addition to IL-9, IL-10, and IL-13, mean levels of IFNγ were shown to be significantly higher in BIA-ALCL seromas. Furthermore, the levels of IL-10, IL-9, and IL-13 were strongly correlated (Figure 1B). IL-10 exceeded 100 pg/mL in 23 of 26 (88%) of malignant seromas but not in any benign effusion. In contrast, IL-6 values were elevated and widely variable in both malignant and benign effusions. Receiver Operating Characteristic (ROC) curve analysis indicated that each of the three cytokines differentiated between BIA-ALCL and benign samples (Figure 1C), with sensitivity, specificity and Youden's J index, respectively, being 0.96/0.83/0.79 for IL-9; 0.88/1.0/0.88 for IL-10, and 0.73/0.97/0.70 for IL-13, therefore giving IL-10 the best predictive diagnostic value. Because of the highest predictive value of IL-10 for BIA-ALCL, a lateral flow assay (LFA) was developed to detect IL-10. A range of detection for IL-10 of 250–10 000 pg/mL was determined by adding known concentrations of recombinant IL-10 protein to benign seroma 69 (Figure S3). Clinical samples were diluted 10× and run on LFA strips containing a test line specific to IL-10. Strips were imaged using a smartphone camera and images were uploaded to a computer. ImageJ (Gel Analyzer tool) was used to obtain intensity profiles for each strip, and test line and control line values were determined as the area under the curve for each peak. The ratio of the test line to the control line (TL/CL) was obtained by dividing the values for each strip. A detailed LFA protocol is provided in Supplementary file 1. IL-10 LFA was able to distinguish 9 BIA-ALCL from 10 benign seromas (Figures 1D and S4.). The dense test lines in seromas 1732 and 1736 correspond to diluted concentrations of 46 767 and 75 323 pg/mL, respectively whereas the faint test line of seroma 61 corresponds to a diluted concentration of 12 575 pg/mL determined by flow cytometry. All malignant samples gave a TL/CL ratio greater than 0.05. (Figure 1E). Our long-term goal is to develop a clinical point of care test for screening and diagnosis of BIA-ALCL in early-stage disease avoiding cytotoxic therapy. To accomplish this goal we aim to develop a multiplex LFA employing highly sensitive CD30 with IL-10. CD30 is not specific for BIA-ALCL because CD30 is expressed by activated lymphocytes and CD30+ cells can be detected in some benign seromas. In contrast, our studies show that IL-10 is less sensitive but more specific for BIA-ALCL at 1:10 dilutions of clinical seromas. We expect increased specificity with retained sensitivity by combining both markers. These findings potentially enable future rapid point-of-care testing for BIA-ALCL by unspecialized personnel at low cost. The amount of fluid (1 mL) is far less than used or required for current practice of histology, immunohistochemistry and/or flow cytometry and clonal analysis.6 The ability to detect BIA-ALCL in 1 mL of fluid will reduce the number of patients exposed to disease progression and death due to financial barriers. Currently, at the time of routine explant, extensive capsule testing required to rule out BIA-ALCL and/or confirm negative margins is not routinely ordered or performed. If capsules requiring this extensive testing could be easily identified by LFA, this would reduce the number of cases where the disease is missed. When an incidental diagnosis of BIA-ALCL is missed at explant, the patient is exposed to disease progression from incomplete capsulectomy, inadequate follow-up, and/or inadequate treatment. This study also provides insight into the pathogenesis and clinical evolution of BIA-ALCL. IL-10 is an immunosuppressive/anti-inflammatory cytokine that may contribute to the long delay (median 9 years) in development of BIA-ALCL following implant. IL-9 and IL-13 are important mediators of allergic inflammation consistent with eosinophils frequently found in BIA-ALCL. In conclusion, this study in the largest cohort of patients to date, shows the diagnostic utility of cytokine analysis for detection of early-stage BIA-ALCL. IL-10 was found to have the highest predictive value for the diagnosis of BIA-ALCL. Lateral flow assay can potentially provide a rapid low-cost point-of-care screening test for BIA-ALCL run by unspecialized personnel, expediting patient care, including patients in underserved areas. Marshall E. Kadin designed the study and wrote the paper. Honghua Hu, Haiying Xu, Kelly Fan, Kristen Brosamer, and Imran Khan performed research. Elena Shklovskaya, Kristen Brosamer, and Mithun Sinha analyzed data. Anand Deva and Richard Willson contributed vital resources. All authors have reviewed and approved the final manuscript. Grant IIT-2018-10408 from Allergan, a subdivision of AbbVie and a grant from the Aesthetic Surgery Research and Education Foundation (ASERF), both to MEK. The data that support the findings of this study are available from the corresponding author upon reasonable request. Data S1. Supplementary methods. Figure S1. Cytology of BIA-ALCL seroma 61 and benign seroma 65. Figure S2. Immunohistochemistry of IL-10, IL-13, and IL-9 in tumor cells. Figure S3. LFAs for recombinant IL-10 protein. Figure S4. IL-10 LFA results for 48 h supernatants of BIA-ALCL lines and additional clinical samples. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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